Project Summary/Abstract Our long-term goal is to understand specific functions of the heterodimeric (PC1cFL) and uncleaved (PC1U) states of Polycystin-1 (PC1) in kidney development, homeostasis, and PKD pathogenesis. This project builds on our previous key findings involving cleavage of PC1 at the GPS motif and investigates molecular and biophysical mechanisms by which cleavage regulates the trafficking and function of PC1. The central hypothesis is that the GPS motif and the adjacent linker form a bipartite force-transduction module that mediates critical functions of PC1 in both the heterodimeric (PC1cFL) and uncleaved (PC1U) states. In PC1cFL, GPS cleavage generates a conformational change of the last ?-strand (?-1) within the GPS motif, which enables ciliary trafficking and function via signal transmittance from the rigid linker to the N-terminal transmembrane domain. In PC1U, the linker transduces cleavage-independent forces. This proposal will use a multi-disciplinary approach to test the functional role of the GPS-linker module. 1) We will test the hypothesis that a tight association of the ?1-strand within the GPS is required for PC1 ciliary trafficking and function, and is disrupted by PKD1-associated mutations. We predict that tight association of this ?-strand within the GPS motif is required to enable PC1 to recruit Rabep1/GGA1/Arl3, traffic to cilia, and induce in vitro tubulogenesis. 2) We will test the hypothesis that high rigidity and short length in the linker is required for PC1 ciliary trafficking and function, and is disrupted by PKD1-associated mutations. We predict that the rigidity of the linker is required to enable PC1 to recruit Rabep1/GGA1/Arl3, traffic to cilia, and induce in vitro tubulogenesis. 3) We will determine the in vivo role and mechanism of the GPS-linker module by generating two Pkd1 knockin mouse models, one with impaired GPS association and the other with a flexible linker. We will examine their kidney phenotypes at development and after birth, and analyze the biogenesis and trafficking of mutant PC1 in proximal and distal nephron cells. The proposed studies will provide novel mechanistic insights into the GPS-linker module and dissect their functional role for the two PC1 forms at kidney development and in different nephron-segments in postnatal periods. These insights should result in therapeutic strategies that restore normal PC1 function in a significant proportion of ADPKD patients by manipulating the GPS-linker module force-transduction process.